DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.     

A family study in transposition of the great vessels and in tricuspid atresia

Summary A group of 186 propositi with transposition of the great vessels (TGV) and 33 propositi with tricuspid atresia (TA) were studied. Data were obtained from hospital records and complemented by questionnaires and in part by personal interview and clinical examination. The main results were among sibs of propositi with TGV an increased incidence of TGV (0.0135±0.0054) and of congenital heart disease in general (0.0495±0.0103) (K=0, r_min=1). Among 92 sibs of propositi with TA only one with patent ductus arteriosus was found. The sex ratio of the propositi with TGV was 1.95. There was also noted a highly abnormal sex ratio among sibs of parents of patients with TGV in part confirming the findings of Knox.A male excess was found in paternal and maternal sibships and was also present in sibships of propositi. Data about seasonal distribution were inconclusive as were data about consanguinity of parents. No parental age or birth order effect was found in either group.This work has been carried out in part during the author's stay at the Genetic Clinic of the Children's Memorial Hospital, Dept. of Pediatrics, Northwestern University Medical School (Prof. D. Y.-Y. Hsia, M.D.). Data have been collected in collaboration with Dr. M. H. Paul, director of the Dept. of Cardiology of the Children's Memorial Hospital, Willis J. Potts Children's Heart Center. The author was supported in part by a grant from the US Public Health Service, National Institutes of Health and a Fulbright Travel Grant.     

Assessment of cutaneous blood flow by using topographical perfusion mapping techniques

The ability of laser Doppler scanning to reproduce the spatialpattern of cutaneous vascular conductance (CVC) in a6.25-cm² area of skin wasevaluated at supine rest (28°C), during thermal stress (cold andheat), and during baroreceptor unloading with 40-mmHg lower bodynegative pressure (LBNP). The spatial pattern of resting CVC wassimilar on 3 different days, varying by 6 ± 3%. During coldstress, 89 ± 2% of the skin area showed a decrease in skin bloodflow (37 ± 2%), whereas heat stress increased CVC in 94 ± 5%of the skin area. During LBNP, the pattern of CVC response was notuniform, and frequency analysis indicated that 47 ± 5% of thepixels showed a reduction in CVC (>1 SE), 28 ± 2% of the skinarea were unaffected, and the remaining 26 ± 5% of the pixels showed some increase in CVC. These data indicate the ability of topographical perfusion mapping to provide quantitative andreproducible information about the spatial distribution of CVC. Inaddition, the site-to-site variability in reflex control of skin bloodflow during LBNP is intriguing and requires more rigorous evaluation.

     

Production of high-titer transmission-defective RNA virus-based episomal vector using tangential flow filtration

In recent years, viral vector based in vivo gene delivery strategies have achieved a significant success in the treatment of genetic diseases. RNA virus-based episomal vector lacking viral glycoprotein gene (ΔG-REVec) is a nontransmissive gene delivery system that enables long-term gene expression in a variety of cell types in vitro, yet in vivo gene delivery has not been successful due to the difficulty in producing high titer vector. The present study showed that tangential flow filtration (TFF) can be effectively employed to increase the titer of ΔG-REVec. Concentration and diafiltration of ΔG-REVec using TFF significantly increased its titer without loss of infectious activity. Importantly, intracranial administration of high titer vector enabled persistent transgene expression in rodent brain.     

High performance flow injection analysis of recombinant protein G

Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous flow injection analysis. Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution. This together with their price and their long-standing time made them attractive for use in flow injection analysis. The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer. The epoxy groups inherent to this base structure can be used for immobilization purposes. In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli. A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.     

Size-frequency analysis of atresia in cycling rats

The purpose of this study was to delineate when, during follicular growth, the alternative developmental pathways leading to ovulation or atresia diverge. By using computerized image analysis techniques, random samples of healthy and atretic follicles in ovaries of cycling rats were subjected to size-frequency analysis. The vast preponderance of atretic follicles were of the early antral size class (approximately 300-350 micron diameter, 800-1000 granulosa cells in the largest cross-section); atretic small follicles (less than 250 granulosa cells in the largest cross-section) were rare. Follicles in early stages of atresia were uncommon in ovaries of animals killed at estrus, but were found with great frequency in ovaries of animals killed the following day (metestrus). These results suggest that, under normal cyclic conditions, there may be only one major branching point during follicular development when growing follicles become susceptible to atresia. The alternative developmental pathways leading to ovulation and atresia may not diverge until the penultimate stage of growth, immediately preceding the final transformation into a preovulatory follicle.     

Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography

The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.     

Development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides

In the present study a HPAEC-PAD method is described that was developed for monitoring the consistency of N-glycosylation during the production and purification of recombinant proteins and monoclonal antibodies. The method successfully separated 18 neutral and sialylated oligosaccharides. Results obtained were compared with MALDI-TOF MS and it was shown that both methods gave similar results. In addition, a method validation was performed showing that the HPAEC-PAD analysis was well suited for the mapping and characterization of oligosaccharides. The method was found to be robust and additionally the precision was significantly better compared to the MALDI-TOF MS method.     

Whole-genome linkage analysis in mapping alcoholism genes using single-nucleotide polymorphisms and microsatellites

There is currently a great interest in using single-nucleotide polymorphisms (SNPs) in genetic linkage and association studies because of the abundance of SNPs as well as the availability of high-throughput genotyping technologies. In this study, we compared the performance of whole-genome scans using SNPs with microsatellites on 143 pedigrees from the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workshop 14. A total of 315 microsatellites and 10,081 SNPs from Affymetrix on 22 autosomal chromosomes were used in our analyses. We found that the results from the two scans had good overall concordance. One region on chromosome 2 and two regions on chromosome 7 showed significant linkage signals (i.e., NPL >or= 2) for alcoholism from both the SNP and microsatellite scans. The different results observed between the two scans may be explained by the difference observed in information content between the SNPs and the microsatellites.     

The efficiency of mapping of quantitative trait loci using cofactor analysis in half-sib design

This simulation study was designed to study the power and type I error rate in QTL mapping using cofactor analysis in half-sib designs. A number of scenarios were simulated with different power to identify QTL by varying family size, heritability, QTL effect and map density, and three threshold levels for cofactor were considered. Generally cofactor analysis did not increase the power of QTL mapping in a half-sib design, but increased the type I error rate. The exception was with small family size where the number of correctly identified QTL increased by 13% when heritability was high and 21% when heritability was low. However, in the same scenarios the number of false positives increased by 49% and 45% respectively. With a liberal threshold level of 10% for cofactor combined with a low heritability, the number of correctly identified QTL increased by 14% but there was a 41% increase in the number of false positives. Also, the power of QTL mapping did not increase with cofactor analysis in scenarios with unequal QTL effect, sparse marker density and large QTL effect (25% of the genetic variance), but the type I error rate tended to increase. A priori, cofactor analysis was expected to have higher power than individual chromosome analysis especially in experiments with lower power to detect QTL. Our study shows that cofactor analysis increased the number of false positives in all scenarios with low heritability and the increase was up to 50% in low power experiments and with lower thresholds for cofactors.     

Selective determination of lithium in biological fluids using flow injection analysis

The use of flow injection analysis to automated extraction methods for the determination of lithium ion utilizing crown ethers or cryptands is demonstrated. The ion-pair extraction of cryptand 211, lithium, and resazurin exhibits a linear range for lithium ion of 70 ppb to 2.1 ppm. This method could tolerate up to 1000 ppm sodium ion. The chromogenic crown ether, 1-(2-hydroxy-5-nitrobenzyl)-1-aza-4,7,10-trioxacyclododecane, exhibits a linear range for lithium ion of 0.3 to 2 ppm. A sodium ion concentration of 230 ppm can be tolerated. Both extraction systems were used in the automated determination of lithium in blood serum and urine. Both methods agreed well with the known and/or atomic absorption values.     

Identification of novel schizophrenia loci by homozygosity mapping using DNA microarray analysis

The recent development of high-resolution DNA microarrays, in which hundreds of thousands of single nucleotide polymorphisms (SNPs) are genotyped, enables the rapid identification of susceptibility genes for complex diseases. Clusters of these SNPs may show runs of homozygosity (ROHs) that can be analyzed for association with disease. An analysis of patients whose parents were first cousins enables the search for autozygous segments in their offspring. Here, using the Affymetrix® Genome-Wide Human SNP Array 5.0 to determine ROHs, we genotyped 9 individuals with schizophrenia (SCZ) whose parents were first cousins. We identified overlapping ROHs on chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 19, 20, and 21 in at least 3 individuals. Only the locus on chromosome 5 has been reported previously. The ROHs on chromosome 5q23.3–q31.1 include the candidate genes histidine triad nucleotide binding protein 1 (HINT1) and acyl-CoA synthetase long-chain family member 6 (ACSL6). Other overlapping ROHs may contain novel rare recessive variants that affect SCZ specifically in our samples, given the highly heterozygous nature of SCZ. Analysis of patients whose parents are first cousins may provide new insights for the genetic analysis of psychiatric diseases.     

Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods

Although the biological importance of sulfated oligosaccharides has been widely recognized, there are only a few reports that describe detailed structures of sulfated N-glycans. This is largely due to the lack of a convenient method to identify structures of sulfated glycans found in low incidence. Here we develop multidimensional high performance liquid chromatography (HPLC) mapping methods for rapid and convenient identification of sulfated N-glycans. By using adequate quantities of sulfated N-glycans derived from LS12 cells, which are transfected with sulfotransferase cDNA, 40 different sulfated glycans have been successfully mapped. Furthermore, we have applied the HPLC data to identification of isomeric products resulting from an enzymatic reaction of N-acetylglucosamine 6-O-sulfotransferase-1 in vitro and revealed that this enzyme preferentially catalyzes sulfation of the GlcNAcbeta1-->2Manalpha1-->3Man branch in a biantennary acceptor.